The overall objectives of this project are to delineate the pathways of hepatic bilirubin transport, to define the mechanisms of microsomal formation of bilirubin glucuronides, and to evaluate the extent and metabolic signifance of bilirubin compartmentation in the liver. Several different approaches are being employed using recently developed probes of bile pigment metabolism (e.g., isotopically labeled bilirubin precursors and glucuronides) applied in a variety of experimental systems (e.g., isolated rat liver perfused with a heme-free, fluorocarbon medium, and microsomal preparations). Specifically, experiments are being performed to define the metabolic pathways of bile pigment derived from both intra- and extrahepatic heme sources, and a multicompartmental model has been designed describing transport kinetics of bilirubin. This model will be tested by additional kinetic studies using bilirubin precursors such as biliverdin. The subcellular localization of individual bilirubin compartments will be examined using subcellular fractionation of hepatocytes and autoradiography. In addition, the enzymatic synthesis of bilirubin mono- and diglucuronide is currently being examined in a non-detergent activated rat liver microsomal system and catalytic properties of UDP-glucuronyltransferase evaluated. Collectively, the information derived from these studies should result in an improved understanding of the regulation of bile pigment metabolism and mechanisms of hepatic transport of bilirubin, other endogenous organic anions and xenobiotics.